CHEMISTRY AND FUNCTION OF DICTYOSTELIUM CALMODULIN

盘基菌钙调蛋白的化学性质和功能

• 批准号: 3277365
• 负责人: MARGARET B CLARKE
• 金额: $ 17.83万
• 依托单位: OKLAHOMA MEDICAL RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-08-01 至 1993-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3277365
• 关键词: Dictyostelium; calmodulin; cell differentiation; cell motility; chemical structure function; chemotaxis; cyclic AMP; density gradient ultracentrifugation; eukaryote; fluorescence microscopy; fungal genetics; genetic manipulation; immunochemistry; immunofluorescence technique; molecular cloning; monoclonal antibody; mutant; myosins; nucleic acid sequence; phagocytosis; pinocytosis; point mutation; protein sequence; radioimmunoassay; spectrometry; tissue /cell culture

Calmodulin is a calcium-binding protein found in all eukaryotic cells; it appears to be responsible for mediating the regulatory effects of calcium on numerous biological processes. The protein has been highly conserved, so its structure and functions can be conveniently examined in lower eukaryotes as well as vertebrate cells. This project would characterize calmodulin from the eukaryotic microorganism Dictyostelium discoideum and explore its roles, particularly in the regulation of motility. Earlier work has revealed that this protein, although functionally similar to vertebrate calmodulins, has several amino acid differences, including the absence of trimethylated lysine residue. Determination of the primary structure will be completed, and the consequences of the amino acid differences will be explored. Interaction with Ca++, troponin I, and vertebrate enzymes will be examined in this regard. Monoclonal antibodies will be raised against Dictyostelium calmodulin; the site (or sites) toward which these antibodies are directed will be determined, and mutants producing calmodulin altered at this site will be sought. Proteins that interact with Dictyostelium calmodulin will be identified and characterized, with emphasis on proteins that may be involved in regulating the structural states or interaction of myosin and actin. The ability of calmodulin to modulate myosin force production through a light chain kinase or other means will be tested directly. Antibodies against Dictyostelium calmodulin, myosin, and selected calmodulin-binding proteins will be used to visualize the localization of these proteins in mitotic and chemotaxing cells; for some of these proteins, monoclonal antibodies will be raised to facilitate purification and to map functional domains. The gene(s) encoding Dictyostelium calmodulin will be isolated and sequenced, as an initial step toward a long range goal of analyzing the function of the protein by genetically modifying it.

钙调蛋白是一种存在于所有真核细胞中的钙结合蛋白；它 似乎负责介导钙的调节作用 对许多生物过程的影响。 该蛋白质高度保守， 因此其结构和功能可以方便地在较低的状态下检查 真核生物以及脊椎动物细胞。 该项目的特点是 来自真核微生物盘基网柄菌的钙调蛋白和 探索其作用，特别是在运动调节方面。 早些时候 研究表明，这种蛋白质虽然在功能上与 脊椎动物钙调蛋白，有几个氨基酸差异，包括 不存在三甲基化赖氨酸残基。 初级的确定 结构将完成，氨基酸的后果 将探讨差异。 与 Ca++、肌钙蛋白 I 和 将在这方面检查脊椎动物的酶。 单克隆抗体 将针对盘基网柄菌钙调蛋白提出；该网站（或多个网站） 这些抗体针对哪些抗体将被确定，并且突变体 将寻求在该位点产生改变的钙调蛋白。 蛋白质 与盘基网柄菌钙调蛋白的相互作用将被鉴定并 特征，重点是可能参与调节的蛋白质 肌球蛋白和肌动蛋白的结构状态或相互作用。 的能力 钙调蛋白通过轻链激酶调节肌球蛋白力的产生 或其他方式直接进行测试。 盘基网柄菌抗体 将使用钙调蛋白、肌球蛋白和选定的钙调蛋白结合蛋白 可视化这些蛋白质在有丝分裂和趋化作用中的定位 细胞；对于其中一些蛋白质，将产生单克隆抗体 促进纯化并绘制功能域图。 基因 编码盘基网柄菌钙调蛋白的蛋白将被分离并测序，作为 迈向分析功能的长期目标的第一步 通过基因改造蛋白质。